A reply to Answers in Genesis regarding
the Apo AI Milano mutation

Introduction:

Glossary

allele: one of several
possible forms of a gene.amino acid: a small organic
molecule used as a building block for proteins.apolipoprotein: the protein component of a
lipoprotein.apolipoprotein AI (Apo-AI): the major protein for forming
HDL
cholesterol lipoproteins; that is, good
HDL.cholesterol: a lipid (fat)
which is an important part of animal cells. It is
transported through the bloodstream as lipoproteins; but in isolation it would
congeal into a solid.dimer: a molecule formed by two identical simpler
units, called monomers.gene: a segment of DNA
which contributes to the function or form of an organism.
Genes mostly work by coding for protein.heterozygote:An organism
that carries two different alleles of
the same gene.homozygote:
An organism that carries two identical alleles of the same gene.lipid: lipids (fats)
are defined as substances which dissolve in alcohol but not
in water. Fats are an important part of all cells, and a
major source of fuel.lipoprotein: a particle
which combines lipids and protein. The proteins allow the
particle to dissolve in water, which means that the lipid
can be transported in the blood stream.LDL,
HDL: low density lipoprotein, high density lipoprotein.
Lipoproteins are characterised by their
density.LDL cholesterol, HDL cholesterol: Cholesterol in LDL
lipoproteins is called "bad" cholesterol, because the LDL
can deposit cholesterol on artery walls, restricting blood
flow.
Cholesterol in HDL lipoproteins is called "good"
cholesterol, because HDL can capture and remove cholesterol
from the arteries and get transported out of the blood
stream through the liver. A good,
simple explanation is provided by the
University of Miami School of Medicine.protein: a large
organic molecule made up of amino acids linked in a
chain.

The
lipid-binding protein called
Apolipoprotein AI (Apo-AI) is the major component of High
Density Lipoprotein (HDL) particles, which play an
important role in
removing cholesterol from cells. In the 1980's an
Italian community was found to have a mutant version of
this protein, Apolipoprotein AI (Milano) (henceforth
referred to as Apo-AIM), and a decreased risk of
arteriosclerosis (clogged arteries), heart attack and
stroke (1). The reduction in risk in
these people has been attributed to the mutant Apo-AIM and
is often used as an example of a beneficial mutation.

Answers in Genesis has a
feedback response (last accessed 19/4/03 [see Note]), which claims that the mutant
provides no evidence for evolution as it has lost
"specificity" (and by implication has lost "information")
and the mutant Apo-AIM is restricted in its ability to
form useful HDL particles. The basis for these claims
appears to be two press releases (2,3), rather than the primary literature.

These press releases are not entirely accurate, and they
appear to have been substantially misinterpreted. Contrary
to what is stated by AIG, Apo-AIM is a
fully functional protein that lowers the risk of
arteriosclerosis and cardiovascular disease by a number of
mechanisms (4). In an experiment where
reconstituted HDL particles made of Apo-AIM were infused into
arteriosclerotic rabbits, the rabbits had fewer and less
extensive plaques (4). There was decreased
aortic cholesterol, and decreased cell proliferation (both
of which improve cell wall flexibility (4)).

While not all of the anti-arteriosclerotic actions of
Apo-AIM are fully understood, we now know of two major
mechanisms involved in its action. Apo-AIM actively
stimulates cholesterol removal from cells (5), and its antioxidant ability also
prevents some of the
inflammatory damage in arteriosclerosis (6). AIG has focused on the antioxidant
activity (possibly because of its prominence in the U.S.
Department of Energy (DOE) press release (2)),
claiming that this represents a loss of
"specificity". In the following sections we will
examine AIG's claims more closely.

Are Apolipoprotein AI-M dimers "of restricted
usefulness"?

In the AIG page it is stated:

"One amino acid has been replaced
with a cysteine residue in an enzyme that normally
assembles high density lipoproteins (HDLs), which are
involved in removing 'bad' cholesterol from arteries. The
mutant form of the enzyme is less effective at what it is
supposed to do, but it does act as an antioxidant, which
seems to prevent atherosclerosis (hardening of arteries).
In fact, because of the added -SH on the cysteine, 70% of
the enzymes manufactured bind together in pairs (called
dimers), restricting their usefulness."

This statement contains a number of inaccuracies and
mistakes. Apo-AI is a lipid-binding protein (not an enzyme)
that forms complexes with other proteins (like Apo-AII) and
lipids to form HDL particles. Apo-AI can self-associate, and
normally forms modest amounts of dimers, trimers and
quadramers as well as being in the monomeric form, and
produces HDL particles in a range of sizes (7). In Apo-AIM the basic amino acid arginine
(R) at position 173 has been
mutated to a sulphur-containing amino acid, cysteine (C; R173C). This results in a
greater ability to form stable dimers than Apo-AI as the
two cysteines can form a chemical bond together via these
sulphur groups (7).

Despite AIG's claim that the formation of dimers results
in "restricting their usefulness" (this claim is not
in either of the press releases), they are in fact fully
functional. Cholesterol removal from cells occurs primarily
through reverse cholesterol transport (RCT). The
first step in RCT is the efflux of un-esterified
cholesterol from cells to suitable acceptors, normally
apolipoproteins (with or without lipids already bound). This
occurs through two distinct mechanisms: a) nonspecific
interaction of lipoprotein acceptors with the cell, and
diffusion of cholesterol from the cell membrane onto the
lipoprotein surface, and b) interaction of lipid-free
apolipoproteins with specific acceptor sites on the cell
surface. Cholesterol then diffuses into the
apolipoproteins. Both of these mechanisms depend on the
structure of Apo-AI.

The Apo-AIM dimers bind to the specific Apo-AI binding
site (lecithin:cholesterol acyltransferase; LCAT)
as efficiently as Apo-AI (5,7) and stimulates cholesterol efflux (5,8). Apo-AIM dimers form
HDL particles as readily as Apo-AI monomers (7), and the HDL formed from Apo-AIM
stimulates cholesterol efflux more efficiently than HDL
formed from Apo-AI monomers (5,8). Apo AIM-containing HDL is also far more
efficient at inhibiting cholesterol esterification by
microsomal acylCoA:cholesterol acyltransferase (ACAT),
than Apo AI-containing HDL (5). This
results in more cholesterol being released from the
membrane for efflux into HDL particles.

Apo-AIM does form a more restricted size range
of HDL, with predominant sizes of 7.8, 12.7 nm (and a rare 10.8 nm form),
whereas Apo-AI forms particles of 7.8, 9.6, 12.7 and
(mostly only seen at high lipid concentrations) 17.6 nm (7). This is in part because, as mentioned
above, Apo-AI can form dimers and higher oligomers, while
Apo-AIM can only form dimers and quadramers. Note again
that normal Apo-AI can and does dimerise, and dimeric
Apo-AI is incorporated into HDL particles just as dimeric
Apo-AIM is. Despite the restricted size range, the HDL
formed by Apo-AIM dimers are functional (5,8,7).
While it was claimed in the DOE press release (2) that dimerisation is responsible for the
HDL deficiency seen in people carrying the Apo-AIM gene,
the dimers are actually more stable than Apo-AI monomers
(9); indeed, part of their ability to
reduce the risk of arteriosclerosis may be due to their
stability, resulting in their hanging round for longer,
mopping up more cholesterol and activating whatever they
activate to inhibit cell proliferation (9). So we can see that Apo-AIM dimers are
not restricted in usefulness, and do form fully functional
HDL particles. These Apo-AIM dimer HDL particles are more
stable than Apo-AI HDL particles (9), and
are better than Apo-AI HDL particles at stimulating
cholesterol efflux (5,8).

Is the antioxidant activity of Apo-AIM
"non-specific"?

While AIG concedes that Apo-AIM has gained a new
function, the antioxidant ability, they contend that this
comes at the expense of specificity.

"Now in gaining an anti-oxidant activity, the enzyme
has lost activity for making HDLs. So the mutant enzyme has
sacrificed a lot of specificity. Since antioxidant activity
is not a very specific activity (a great variety of simple
chemicals will act as antioxidants), it would seem that the
net result of this mutation has been a huge loss of
specificity, or, in other words, information. This is
exactly as we would expect with a random change."

As we have seen, Apo-AIM has not lost the ability to
make HDLs, so it has not sacrificed specificity. Indeed, as
Apo-AIM HDL particles are more effective at promoting
cholesterol removal from cells, one could reasonably claim
that there has been an increase in specificity. However, is
the antioxidant activity of Apo-AIM non-specific?
Antioxidant activity is possessed by a number of small
molecules, but so is protein hydrolysis (catalyzed by small
molecules such as the amino acid serine), esterification
and just about all important enzyme activities. What
matters to an extent is if there is a specific sequence
that binds a substrate to deliver it to the antioxidant
amino acid (in the same way the catalytic site sequence in
a protein hydrolysis enzyme delivers the substrate to
catalytic serine). Is the antioxidant activity of Apo-AIM
substrate and sequence specific?

The answer is yes. The antioxidant effect is sequence
dependent. The Milano mutation (R173C) is much more
effective at inhibiting oxidation of lipids than another
mutation, the Paris mutation (R151C) (6).
There is also substrate specificity. Neither mutation can
quench superoxide anions in aqueous solution (6), suggesting that the R->C mutation
does not generate a generic antioxidant, but rather a
specific targeted antioxidant that will only work in a
specific way. Neither mutation is capable of preventing
oxidation of a control protein (cytochrome c) (6) so the mutation is specific for lipid
substrates. Further studies (10) have
indicated that small peptides derived from these regions
(amino acids 167-R173C-184 and 145-R151C-162) also retain
the specificity for lipids, indicating that it is not
simply the presence of the cysteine, but rather the
position of the cysteine within the structural constraints
of the protein, that confer the healthy antioxidant
properties. Thus we can see that the antioxidant properties
are specific, in the sense that they are substrate and
sequence dependent.

The AIG response also implies that the dimerisation of
Apo-AIM is irreversible. In fact, cysteine dimerisation is
a readily reversible reaction, and is used as a control
mechanism in several proteins (for example HSP33 (11)). Dimer formation is reversible by
exposure to reducing environments (6),
and while binding to arteriosclerotic plaques puts the
Apo-AIM HDL in an oxidizing environment, the Apo-AI HDL
also binds to a number of other cellular environments where
there are coupled reduction-oxidization reactions which
specifically reverse cysteine dimerisation. Furthermore,
disulphides (R-S-S-R, where R is an organic compound or
protein) are also antioxidants in certain circumstances.
R-S-S-R compounds are quite capable of being sinks for
electron oxidation (12).

Does the Apo-AIM mutation represent a loss of
information?

The AIG page claims that "information-increasing
mutations" are "required" for evolution. The
concept of "information" is a problematic one in biology,
as most measures only imperfectly capture key aspects of
genetic change. Biologists prefer to think in terms of gene
number and gene/protein function. While an increase in gene
number and function is not required (parasites which have
lost genes do quite nicely), increase in gene number has
occurred. Certainly the average vertebrate has more protein
coding genes than worms or insects, and these in turn have
more protein coding genes than unicellular yeast. The basis
of this increase is largely via duplication of pre-existing
genes. For example, one of the major differences between
vertebrates and worms, and worms and yeasts, is an increase
in the numbers of modified copies of a class of enzymes
called tyrosine kinases. By most measures of "information"
a vertebrate with 30,000 genes in its genome has more
information than a yeast with a mere 6,000 or so genes, and
the role of gene duplication in this rise is well
understood (13).

AIG uses another definition of "information," equating
it with "specificity." This was originally coined by Dr
. L. Spetner, and is related to the number of
substrates an enzyme binds (the fewer substrates, the more
specific the enzyme is and the more "information" it has
(14)). Applying this measure to
non-enzymes is not entirely straightforward. With this
measure, it is claimed that random mutations do not
increase "information" in a protein. In the case of
Apo-AIM, AIG claims that the mutant apolipoprotein has lost
specificity as it has lost (or restricted) the ability to
form HDL particles, and the antioxidant ability of Apo-AIM
is "non-specific". We have seen that in fact Apo-AIM has
not lost the ability to form HDL particles, and that these
HDL particles that are formed bind to specific acceptor
sites and are more effective at promoting cholesterol
efflux than normal HDL particles. We have also seen that
the Apo-AIM antioxidant ability is both sequence and
substrate specific. Thus Apo-AIM has not lost "information"
by AIG's own measures. If anything it has gained Spetner
"information".

Are homozygous Apo-AIM mutations lethal?

In addition, AIG suggests that since only heterozygotes of the Apo-AIM
mutation have been identified so far, "the homozygote (both genes the same) A-I
Milano mutation is lethal." Contrary to AIG's claim, this
finding is unsurprising given the present rarity of the
Milano mutation. The early work studying the heterozygous
carriers of this mutant allele identified only 33
individuals, after genetic testing of all inhabitants in an
isolated northern Italian village (about 1000 people, 15). In the gene pool of this village,
where the mutant allele originated and which has an
extremely high concentration of Apo-AIM mutants relative to
other human populations, the Milano allele has a frequency
of only 1.65% (33 mutant alleles out of 2000 total alleles
of this gene). Assuming that those individuals are mating
randomly, it would be somewhat surprising to find a
homozygous individual in a population of 1000 since we
expect to find a homozygote at a frequency of about 1/3700
(the chance of a homozygote is equivalent to 0.0165
squared). However, all 33 of these individuals are known to
be descendents of one original 18th century couple carrying
the Milano mutation. Humans mate with relatives much less
than random, and therefore finding a homozygote in this
population of carriers is highly improbable. Thus, based
upon our current knowledge of the allelic distribution,
there is no reason to suspect that homozygous Milano
mutations are lethal.

Furthermore, transgenic mice have been made that are
homozygous for the human Apo-AI Milano gene, and they are
healthy (16). In fact, the results
indicate that there is a dose responsive benefit for the
allele - one mutant allele is better than none, but two are
best. The physiology and biochemistry of these transgenic
mice (both homo- and heterozygotes) are extremely similar to
that found in humans. These facts prompted the authors to
conclude that the transgenic Apo-AIM mice are an excellent
experimental model system for studying the effects of this
beneficial mutation.

Conclusions:

AIG claims that the Apo-AIM mutation, which produces a
reduction in risk from heart attack and stroke, results in
a loss of specificity. However, these claims are incorrect.
Instead, Apo-AIM is 1) of a more complex tertiary structure
2) more stable and 3) activates cholesterol efflux more
effectively than Apo-AI. Furthermore, Apo-AIM has an
antioxidant activity not present in Apo-AI that is sequence
and substrate specific. Thus, far from a loss of
specificity, Apo-AIM represents a gain of specificity and
"information" by AIG's own measures. Contrary to AIG's
suggestion, all current evidence indicates that the Apo-AIM
mutation is beneficial for its carriers, whether
heterozygous or homozygous.

Note:

AIG has been made aware of some of the errors in its
feedback page (as accessed on 17/3/03); after
correspondence with Dr. Pirie-Shepherd they have made
several modifications (accessed on 23/3/03) but have not
acknowledged Dr. Pirie-Shepherd. Nor have they made it
clear that the enthusiastic endorsement of the feedback
item was made to the previous version.

"70% of the enzymes manufactured bind together in
pairs (called dimers) and are useless." has
become"70% of the enzymes manufactured bind together in pairs
(called dimers), restricting their usefulness."

"..[T]he enzyme has lost activity for making HDLs. So
the mutant enzyme has sacrificed a lot of specificity."
has become"..[T]he enzyme has lost activity for making HDLs. So
the mutant enzyme has sacrificed specificity."

As we have seen these changes do not substantially
ameliorate the errors, or address the the criticisms in Dr.
Pirie-Shepherd's original emails, nor the criticisms in
this essay.

AIG also removed Dr. Pirie-Shepherd's email to the
original feedback response, which pointed out some errors
in assumptions about the reversibility of the dimerisation
process.